Many pathogens, including viruses such as HIV-1 and HIV-2, influenza, hepatitis A/B/C, human papillomavirus (HPV), and dengue, as well as parasites such as malaria and trichinella, can readily alter the amino acid sequence within particular protein epitopes. In view of this behavior and for other purposes, synthetic peptide vaccines are increasingly being explored as alternatives to attenuated or inactivated vaccines. By selection of only those epitopes that confer an effective immunity, epitopes responsible for deleterious immune responses, such as enhancement of disease or T-cell suppression, can be excluded from candidate vaccines. Additionally, as they are chemically defined and lack any infectious material, they pose minimal health risks. Finally, unlike live attenuated vaccines which must be transported and stored at defined, refrigerated temperatures, peptide vaccines are relatively stable and do not require refrigeration, thus making distribution far easier and less costly.
Currently, synthetic peptide vaccines are being evaluated for protection against bacteria, parasites, and viruses. Bacterial epitope vaccines include those directed against Cholera and Shigella. A synthetic vaccine against malaria has undergone phase I and phase II clinical trials in humans. Influenza and hepatitis B viruses represent two viral systems in which synthetic vaccines look especially promising, and there has been much recent interest in synthetic vaccines against human immunodeficiency virus (HIV).
Despite recent advances, synthetic peptide vaccines have been unable to account for envelope or surface protein variability. Several groups have attempted in the past to account for epitope variability using a variety of approaches. One approach to address epitope variability has been described by Tam in U.S. Pat. No. 5,229,490 (Jul. 20, 1993). This process involves conjugating several similar or different epitopes to an immunogenic core by using lysine functional groups and glycine linkers (called dendritic polymers). This process is referred to as a multiple antigen peptide system (MAPS). While highly immunogenic, HIV-based MAPS have not proven to induce broadly reactive antibodies that can recognize divergent strains of virus (Nardelli et al. (1992) J Immunol 148:914–920).
Another early approach involved the identification of ‘mimotopes,’ which are randomly generated sequences which mimic antigenic epitopes (Lenstra et al. (1992) J Immunol Methods 152:149–157). Using this approach, degenerate oligonucleotides are inserted into bacterial expression vectors, resulting in an expression library of random peptides 6–8 amino acids in length. Those peptides that mimic antigenic epitopes are identified using sera (containing antibodies) from animals or individuals infected with the pathogen of interest. Indeed, this general approach has been used to identify mimotopes that are recognized by sera from HCV-infected individuals (Prezzi et al. (1996) J Immunol 156:4504–4513). However, the peptides are randomly selected and there is a necessity to acquire and analyse sera from infected subjects in order to formulate the mimotope composition.
A further disadvantage to prior art approaches requiring sera from infected individuals is that many infected individuals do not manage to create appropriate anti-pathogen antibodies. Thus, selecting peptides of interest using patient sera could potentially lead to the omission of important antigenic peptides that mimic epitopes against which infected individuals have been unable to mount an immune response.
Using the SIV:rhesus macaque model for HIV infection of humans a, SIV envelope glycoprotein B cell neutralization and T cell epitope has been described and a synthetic immunogen was designed and synthesized based on the hypervariable and highly antigenic epitope of the SIVmac142 envelope glycoprotein (gp130) (Anderson et al. (1994) Vaccine 12:736–740). This synthetic immunogen consisted of a mixture of peptides representing permutations of amino acid substitutions found in SIV envelope gene sequences. Thus, the synthetic immunogen collectively represented all the in vivo variability observed for this particular epitope. Immunogenicity of this synthetic immunogen was evaluated, and it was shown to induce enhanced amounts of antibodies in immunized rhesus macaques with binding to native biological SIV. Furthermore, it enhanced immunoreactivity to divergent epitope analogs. In this and a subsequent publication (Meyer et al (1998) AIDS Res Human Retro 14:751–760) it was demonstrated that this approach could account for epitope variability.
In recent years it has become clear that T cells are degenerate in their recognition of peptide antigens. This discovery has raised concerns that peptides from some foreign antigens may mimic some self antigens and inadvertently lead to the activation of autoreactive T cells and the onset of autoimmune disease. Consequently, there would be a risk of autoimmune disease associated with immunization of animals or human with a mixture of randomly synthesized peptides. Mimotope processes (Lenstra et al. (1992) J Immunol Methods 152:149–157) select a subset of peptides for immunization from a mixture of randomly synthesized peptides using sera from infected animals or humans, and thus reduces the risk of autoimmune disease. However, the synthetic immunogen formulations described above (Anderson et al. (1994) Vaccine 12:736–740; Meyer et al (1998) AIDS Res Human Retro 14:751–760) do not contain completely random mixtures of peptides, as the peptides generated are based on the addition of only a few of the possible 20 amino acids at only some steps of the synthesis reactions. Nonetheless, synthetic immunogenic formulations such as those described in the prior art may contain over 8,000 different peptide antigens. While this process led to an immunogen which evoked broadly reactive immunity, the formulation was too complex to characterize biochemically or immunologically, and immunization of humans which such a compound would carry with it a significant risk of autoimmune disease. Further, without full compositional analysis, regulatory approval of a mixed peptide composition is not likely to be obtained. Full compositional analysis for a mixed peptide composition having hundreds or thousands of different peptides would be extremely time-consuming, and is unlikely to be cost-effective.
Many of the previously described synthetic immunogenic formulations are mixtures of tens of thousands of different peptides. Given that T cells are degenerate in their recognition of foreign antigens, mixtures of peptides this complex pose the risk of containing peptides which mimic self antigens, which upon immunization could induce a pathogenic autoimmune response. Moreover, the complexity of previous synthesis schemes made it difficult if not impossible to chemically define and assess the quality of multiple individual preparations of the same composition.
On this basis, there is a need for a new process for the design of an immunogenic peptide mixture resulting in a less complex formulation than those described in the prior art, while retaining optimum immunogenicity. Such a new process would allow such mixtures to be prepared and analysed for human use. Further, there is a need for development of assays to be run on such a preparation in order to ensure the integrity and antigenicity of the mixture formed in the synthesis reaction.